1. Field of the Invention
The invention, in the field of molecular and cell biology, relates to a novel method, based on direct expression cloning, for identifying target proteins capable of binding to and/or serving as substrates for receptor or cytoplasmic tyrosine kinases. The invention also relates to novel proteins identified using this method.
2. Description of the Background Art
A variety of polypeptide growth factors and hormones mediate their cellular effects by interacting with cell surface receptors and soluble or cytoplasmic polypeptide containing molecules having tyrosine kinase enzymatic activity (for review, see Williams, L. T. et al., Science 243:1564-1570 (1989); Ullrich, A. et al., Cell 61:203-212 (1990); Carpenter, G. et al. J. Biol. Chem. 265: 7709-7712 (1990)). The interaction of these ligands with their receptors induces a series of events which include receptor dimerization and stimulation of protein tyrosine kinase activity. For the epidermal growth factor receptor (EGFR) as wellxe2x80x94as other receptors with tyrosine kinase activity, such as the platelet derived growth factor receptor (PDGFR), kinase activation and receptor autophosphorylation result in the physical association of the receptor with several cytoplasmic substrates (Ullrich et al., supra).
Two substrates for the EGFR kinase have now been definitively identified in living cells: (a) the phosphatidylinositol specific phospholipase C-xcex3 (PLC-xcex3) and (b) the GTPase activating protein (GAP), a protein which may be in the effector loop of the ras protein (Margolis, B. et al. Cell 57: 1101-1107 (1989); Meisenhelder, J. et al. Cell 57: 1109-1122 (1989); Molloy, C. J. et al. Nature 342: 711-714 (1989); Wahl, M. I. et al. J. Biol. Chem. 265: 3944-3948 (1990); Ellis, C. et al. Nature 343: 377-381 (1990); Kaplan, D. R. et al. Cell 61: 121-133 (1990)).
Similarly, activated PDGFR was shown to tyrosine phosphorylate, and to become associated with PLC-xcex3, GAP, and cellular tyrosine kinases such as pp60src (Gould, K. L. et al., Molec. Cell. Biol. 8:3345-3356 (1988); Meisenhelder, J. et al., Cell 57:1109-1122 (1989); Molloy, C. J. et al., Nature 342:711-714 (1989); Kaplan, D. R. et al., Cell 61:121-133 (1990); Kazlauskas, A. et al., Science 247:1578-1581 (1990); Krypta, R. M. et al., Cell 62:481-492 (1990); Margolis, B. et al., Science 248:607-610 (1990)). While the exact sites responsible for the association of EGFR with either PLC-xcex3 or GAP have not been completely clarified, recent work has begun to identify regions on both the substrate and receptor which contribute to the association.
SH2 (src homology 2) domains appear to be the regions responsible for the association of several tyrosine kinase substrates with activated growth factor receptors. SH2 domains are conserved sequences of about 100 amino acids found in cytoplasmic non-receptor tyrosine kinases such as pp6Osrc, PLC-xcex3, GAP and v-crk (Mayer, B. J. et al., Nature 332:272-275 (1988); Pawson, T. Oncogene 3:491-495 (1988)). While having distinct catalytic domains, all these molecules share conserved SH2 and SH3 (src homology 3) domains and the ability to associate with receptors with tyrosine kinase activity (Anderson, D. et al., Science 250:979-982 (1990)).
Tyrosine kinase activation and receptor autophosphorylation are prerequisites for the association between growth factor receptors and SH2 domain-containing proteins (Margolis, B. et al., Mol.
Cell. Biol. 10:435441 (1990); Kumjian et al., Proc. Natl. Acad. Sci. USA 86:8232-8239 (1989); Kazlauskas, A. et al., Science 247:1578-1581 (1990)). In particular, the carboxyterminal (C-terminal) fragment of the EGFR, which contains all the known autophosphorylation sites, binds specifically to the SH2 domains of GAP and PLC-xcex3 (see below). Hence, a major site of association exists between the SH2 domain of these substrate proteins and the tyrosine phosphorylated C-terminal tail of the EGFR.
With the recognition that binding to the activated tyrosine kinase receptor is conserved among several substrate proteins, efforts to identify additional substrates which share these properties have been undertaken. Target proteins which bind to activated receptors have been identified by analysis of proteins that co-immunoprecipitate with growth factor receptors, or that bind to receptors attached to immobilized matrices (Morrison, D. K. et al., Cell 58:649-657 (1989); Kazlauskas, A.-et al., EMBO J. 9:3279-3286 (1990)). While the identity of some of these proteins is known, several others detected utilizing these approaches have not been fully characterized. Moreover, it is possible that rare target molecules which interact with activated receptors have not been detected due to the limited sensitivity of these techniques; the actual stoichiometry of binding may be low, and the detergent solution necessary to solubilize proteins may disrupt binding.
Conventional approaches to isolate and clone these proteins have been arduous, requiring the use of large quantities of tissue or cells lines to purify sufficient amounts of protein for microsequence analysis and subsequent conventional CDNA cloning. Therefore, a need for new approaches for the cloning and subsequent isolation and identification of these proteins is recognized in the art.
It is an object of the present invention to overcome the deficiencies of the related art.
It is also an object of the present invention to understand and gain control over the regulation of cell growth and oncogenesis by providing the ability to identify target proteins for tyrosine kinases, including both receptor and cytoplasmic tyrosine kinases in eukaryotic organisms.
It is a further object of the present invention to provide a novel expression/cloning system for the rapid cloning of target proteins which bind tyrosine kinase proteins which are present intracellularly and in cell receptors of eukaryotes. The cloning method is based on the ability of a certain class of substrates to bind specifically to the tyrosine-phosphorylated carboxyterminus (C-terminus) of the proteins having tyrosine kinase activity. Non-limiting examples include proteins that bind at least one of cytoplasmic and receptor tyrosine kinases, such as a receptor tyrosine kinase found in epidermal growth factor receptor (EGFR) (see Example VI, below).
Another object of the present invention is to provide a method of cloning tyrosine kinase target proteins, which method important advantages over conventional cloning methods, including avoidance of the laborious and costly task of purifying potential target proteins for microsequencing analysis.
Another object of the present invention is to provide a method for identifying receptor target molecules having tyrosine kinase activity whose association with activation receptors could not otherwise be detected using conventional techniques.
Another object of the present invention is to provide for the identification of structurally or functionally related proteins which, though only weakly homologous at the nucleic acid level, are similar in their property of binding to activated receptors with tyrosine kinase activity, which latter ability is important since conventional screening methods used to identify related genes are typically based on low stringency nucleic acid hybridization. Conventional hybridization-based screening would not have been successful in cloning and identifying such tyrosine kinase target proteins of the present invention, exemplified as non limiting examples as GRB-1, GRB-2, GRB-3, GRB-4, GRB-7 or GRB-10, because of their lack of similarity at the DNA level.
Another object of the present invention is to provide a method for identifying compounds that disrupt or inhibit the interaction between activated tyrosine kinase molecules and those proteins (e. adaptor proteins) they bind.
The methods of the present invention take advantage of the discover that the C-terminus of the EGFR protein in which the tyrosine residues are phosphorylated can bind substrates as described herein. By creating a labelled polypeptide which substantially corresponds to at least a portion of phosphorylation domain of a tyrosine kinase, a probe is provided having at least one phosphorylated tyrosine. Such a probe can be used to detect, identify and/or purify target proteins from solutions or as part of screening of CDNA expression libraries from eukaryotic cells or tissues. Such tyrosine kinase target proteins, discovered according to the present invention, ar termed xe2x80x9cGRBxe2x80x9d (for Growth factor Receptor Bound) for the initial receptor tyrosine kinases used, but which target proteins are not limited to growth factor receptors. Accordingly, GRBs of the present invention include target proteins for any eukaryotic tyrosine kinase which are provided according to the present invention.
The novel cloning methodology of the present invention has been designated, xe2x80x9cCORTxe2x80x9d (for Cloning Of Receptor Targets), and may also be applied to detecting, identifying, cloning or purifying target proteins for any tyrosine kinase, such as a soluble, cytoplasmic or receptor tyrosine kinase.
The method of the present invention is proposed as a novel approach having both generality and rapidity for the identification and cloning of target molecules for tyrosine kinases.
The present invention is thus directed to a method for detecting a target protein in solution, which is a target of a receptor or cytoplasmic tyrosine kinase, the target protein being capable of binding to at least a portion of a tyrosine-phosphorylated polypeptide of the receptor or cytoplasmic tyrosine kinase, the method comprising:
(a) contacting the solution (as a cell, an extract thereof, a lysate thereof, or a supernatant thereof) with a solid phase carrier, causing the binding of the protein to the carrier to provide a carrier-bound target protein;
(b) incubating the carrier-bound target protein with the tyrosine-phosphorylated polypeptide, which has been detectably labeled, allowing the polypeptide to bind to the carrier-bound protein;
(c) removing materials not bound to the carrier-bound target protein;
(d) detecting the presence or measuring the amount of the tyrosine-phosphorylated polypeptide bound to the carrier,
thereby quantitatively or qualitatively detecting the target protein in said solution.
In one embodiment, the receptor or cytoplasmic tyrosine kinase is any eukaryotic tyrosine kinase (e.g., epidermal growth factor receptor, a platelet-derived growth factor receptor, or a fibroblast growth factor receptor, pp60v-src, pp160gag-abl, pp130gag-fps, pp59c-fyn, PDGF receptor B, CSF-1 receptor, pp150c-fms, pp150v-fms, Insulin Receptor, IGF-1 receptor, pp68gag-ros, PLC-xcex3, middle t-pp60s-src middle t-pp62c-yes, and the consensus sequences EEEEEY(PO4)MPMXX (SEQ. ID No: 11), EEEEEY(PO4)VPMXX (SEQ ID NO:12), DDDDDY(PO4)MPMXX (SEQ ID NO:13), and DDDDDY(PO4)VPMXX (SEQ ID NO:14) or a phosphorylatable fragment thereof, preferably a polypeptide of about 10 to 250 amino acid residues, more preferably 10 to 40 or 15 to 50 residues, wherein the polypeptide is produced recombinantly, synthetically or by enzymatic digestion of a purified tyrosine kinase molecule.
This method is preferably performed using a prokaryotic cell, most preferably a bacterial cell such as E. coli. The cell may also be eukaryotic, such as a yeast or a mammalian cell.
Preferably, the phosphorylated polypeptide is detectably labeled.
The solid phase carrier can be any material which can be used to bind a target protein for a tyrosine kinase. The carrier may preferably be a nitrocellulose membrane, such as to which are transferred proteins released for lysed bacterial cells when a library is being screened.
The present invention also provides a method for mapping to a eukaryotic, such a mammalian, human, reurine, or other eukaryotic chromosome a gene encoding a protein which is capable of binding to a tyrosine phosphorylated polypeptide portion of a receptor or cytoplasmic tyrosine kinase molecule, the method comprising:
(a) infecting a host or host cells which a eukaryotic gene expression library;
(b) detecting a clone expressing the protein using a method as described above;
(c) sequencing the DNA of the clone; and
(d) mapping the sequence to a eukaryotic chromosome.
The present invention is also directed to a polypeptide probe useful in the detection of the expression of a protein capable of binding to a tyrosine-phosphorylated polypeptide portion of a receptor or cytoplasmic tyrosine kinase. The probe comprises an amino acid sequence derived from the tyrosine-phosphorylated portion of the receptor or cytoplasmic molecule, or a functional derivative thereof, lacks the tyrosine kinase domain, and the sequence must contain at least one phosphotyrosine residue, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 phosphotyrosines. The probe should be detectably labeled with known labels. A preferred probe has between about 10 and 250 amino acid residues, preferably 10-35, 16-30, 21-35, 15-35, or 20-40 residues.
A probe of the present invention is useful for detecting target proteins for receptor or cytoplasmic tyrosine kinases including but not limited to, epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor (PDGFR), fibroblast growth factor receptor (FGFR), colony stimulating factor-1, (CSF-1), insulin receptor, phospholipase C-xcex3 (PLC-xcex3) and insulin like growth factor-1 (IGF-1), pp60v-src, pp160gag-abl, pp130gag-fps, pp59c-fyn, PDGF receptor B, CSF-1 receptor, pp150c-fms, pp150v-fms, EGF receptor, IGF-1 receptor, pp68gag-ros, PLC, middle t-pp60c-src middle t-pp62c-yes, and the consensus sequence EEEEEY(PO4)MPMX (SEQ. ID NO:11), EEEEEY(PO4)VPMXX (SEQ ID NO:12), DDDDDY(PO4)MPMXX (SEQ ID NO:13), and DDDDDY (PO4)VPMXXX. (SEQ ID NO: 14) or a phosphorylatable fragment thereof, e.g., as described Cantley et al., Cell 64:281-302 (1991) or Ullrich and Schlessinger, Cell 61:203-312 (1990), which references are entirely herein incorporated by reference.
The present invention also includes a method for preparing the above probe, comprising
(a) providing the receptor or cytoplasmic tyrosine kinase, or a recombinantly, enzymatically or synthetically produced fragment thereof wherein the receptor or cytoplasmic tyrosine kinase, or fragment thereof, has both a tyrosine kinase domain and a tyrosine-phosphorylated domain, the tyrosine-phosphorylated domain including at least one tyrosine residue capable of being phosphorylated by the tyrosine kinase;
(b) incubating the receptor or cytoplasmic tyrosine kinase, or fragment, with detectably labeled adenosine triphosphate under conditions permitting phosphorylation of the tyrosine residue, causing phosphorylation of the tyrosine residue thereby producing the probe. In a preferred embodiment, the method includes the step of:
(c) additionally treating the phosphorylated receptor or cytoplasmic tyrosine kinase molecule with an agent capable of cleaving the molecule between the tyrosine kinase domain and the tyrosine-phosphorylated domain.
A preferred cleaving agent is cyanogen bromide.
In another embodiment, the above method involves a genetically engineered receptor-like derivative which is a polypeptide encoded by a DNA molecule comprising a DNA sequence encoding tyrosine kinase, linked to a DNA sequence encoding a selective enzymatic cleavage site, linked to a DNA sequence encoding the tyrosine-phosphorylated domain, and wherein the agent is an enzyme capable of cleaving at this cleavage site. Preferred enzymes are Factor Xa and thrombin.
Also provided is a method for purifying from a complex mixture a protein which is capable of binding to a tyrosine-phosphorylated polypeptide portion of a receptor or cytoplasmic tyrosine kinase molecule, the method comprising:
(a) contacting the complex mixture with a solid phase carrier to which a probe is bound, allowing the protein to bind to the probe;
(b) removing materials not bound to the carrier; and
(c) eluting the bound protein from the carrier,
thereby purifying the protein.
The present invention is also directed to a GRB protein of at least 10 amino acids, including any range of value up to its entire native or mature length. The present invention, in one embodiment, provides a protein, GRB-1, having the amino acid sequence shown in FIG. 4 (SEQ ID NO:5). The invention also includes polypeptides having an amino acid sequence substantially corresponding to the amino acid sequence of a protein, GRB-2 which includes the amino acid sequence shown in FIGS. 26A-26C (SEQ ID NO:6). The invention also includes polypeptides having an amino acid sequence substantially corresponding to the amino acid sequence of a protein, GRB-3, which includes the amino acid sequence shown in FIGS. 34A-34C (SEQ ID NO:8). The invention also includes polypeptides having an amino acid sequence substantially corresponding to the amino acid sequence of a protein, GRB-4, which includes the amino acid sequence shown in FIGS. 35A-35B (SEQ ID NO:9). The invention also includes polypeptides having an amino acid sequence substantially corresponding to the amino acid sequence of a protein, GRB-7, which includes the amino acid sequence shown in FIGS. 36A-36G (SEQ ID NO:10). The invention also includes polypeptides having an amino acid sequence substantially corresponding to an amino acid sequence of a protein, GRB-10, which includes the amino acid sequence shown in FIG. 38 (SEQ ID NO:18).
The invention is also directed to a DNA or RNA molecule encoding a polypeptide having at least a 10 amino acid sequence substantially corresponding to the amino acid sequence of at least one of GRB-1, GRB-2, GRB-3, GRB-4, GRB-7 or GRB-10 proteins. Included are DNA molecules encoding functional derivatives of these proteins. When the DNA molecule naturally occurs, it is substantially free of the nucleotide sequences with which it is natively associated. The DNA molecules of this invention may be expression vehicles, such as plasmids.
Also provided is a host transformed with each of the above DNA molecules.
The present invention also includes a process for preparing a target protein substantially corresponding to the amino acid sequence GRB-1, GRB-2, GRB-3, GRB-4, GRB-7 or GRB-10 protein, comprising:
(a) culturing a host comprising a recombinant nucleic acid having a nucleotide sequence encoding the target protein under culturing conditions such that the target protein is expressed in recoverable amounts; and
(b) recovering the protein from the culture.